Production of Functional Buttermilk and Soymilk Using Pediococcus Acidilactici BD16 (Alad+)

molecules Article Production of Functional Buttermilk and Soymilk Using Pediococcus acidilactici BD16 (alaD+) Anshula Sharma 1, Masafumi Noda 2, Masanori Sugiyama 2, Ajaz Ahmad 3 and Baljinder Kaur 1,* 1 Systems Biology Laboratory, Department of Biotechnology, Punjabi University, Patiala 147002, India; [email protected] 2 Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi, Minami-ku, Hiroshima 734-8553, Japan; [email protected] (M.N.); [email protected] (M.S.) 3 Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] * Correspondence: [email protected] Abstract: Functional foods or drinks prepared using lactic acid bacteria (LAB) have recently gained considerable attention because they can offer additional nutritional and health benefits. The present study aimed to develop functional drinks by the fermentation of buttermilk and soymilk prepara- tions using the Pediococcus acidilactici BD16 (alaD+) strain expressing the L-alanine dehydrogenase enzyme. LAB fermentation was carried out for 24 h and its impact on the physicochemical and quality attributes of the fermented drinks was evaluated. Levels of total antioxidants, phenolics, flavonoids, and especially L-alanine enhanced significantly after LAB fermentation. Further, GC-MS- based metabolomic fingerprinting was performed to identify the presence of bioactive metabolites such as 1,2-benzenedicarboxylic acid, 1-dodecene, 2-aminononadecane, 3-octadecene, 4-octen-3-one, acetic acid, azanonane, benzaldehyde, benzoic acid, chloroacetic acid, colchicine, heptadecaneni- Citation: Sharma, A.; Noda, M.; trile, hexadecanal, quercetin, and triacontane, which could be accountable for the improvement of Sugiyama, M.; Ahmad, A.; Kaur, B. organoleptic attributes and health benefits of the drinks. Meanwhile, the levels of certain undesirable Production of Functional Buttermilk metabolites such as 1-pentadecene, 2-bromopropionic acid, 8-heptadecene, formic acid, and propionic and Soymilk Using Pediococcus acidilactici BD16 (alaD+). Molecules acid, which impart bitterness, rancidity, and unpleasant odor to the fermented drinks, were reduced 2021, 26, 4671. https://doi.org/ considerably after LAB fermentation. This study is probably the first of its kind that highlights the + 10.3390/molecules26154671 application of P. acidilactici BD16 (alaD ) as a starter culture candidate for the production of functional buttermilk and soymilk. Academic Editors: Warren McNabb and Daniel Rico Keywords: bioactive metabolites; functional drinks; L-alanine; LAB fermentation; metabolomic fingerprinting; Pediococcus acidilactici; starter culture bacteria Received: 27 June 2021 Accepted: 30 July 2021 Published: 2 August 2021 1. Introduction Publisher’s Note: MDPI stays neutral In recent years, mounting public awareness regarding diet-associated health issues has with regard to jurisdictional claims in led to the development of natural and healthy functional foods or dietary items. Functional published maps and institutional affil- foods or drinks provide additional nutrients and energy to beneficially modulate metabolic iations. functions of the body and reduce the risks associated with certain diseases [1]. Functional soymilk is a milky fluid obtained from the overnight soaked and mashed soybeans, which contains many bioactive components that can reduce the pathophysio- logical responses associated with gut disorders to improve human health [2]. Soy-based Copyright: © 2021 by the authors. foods or drinks are inexpensive and rich nutritional supplements for vegetarian and Licensee MDPI, Basel, Switzerland. lactose-intolerant people. Previous studies have also reported that these are abundant This article is an open access article in isoflavones and high-quality proteins that help in reducing the risks of Alzheimer’s distributed under the terms and disease, atherosclerosis, cancer, cardiovascular and hepatic disorders, diabetes, irregular conditions of the Creative Commons Attribution (CC BY) license (https:// menstrual cycles and menopausal syndromes, and premature aging. Moreover, they also creativecommons.org/licenses/by/ assist in immunomodulation, neuroprotection, weight management, and enhanced athletic 4.0/). performance [3–5]. The aglycone form of isoflavones is preferred over the glycosidic form Molecules 2021, 26, 4671. https://doi.org/10.3390/molecules26154671 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 4671 2 of 25 due to its enhanced bioavailability and health-imparting attributes. Thus, fermentation of soymilk with a suitable LAB strain is suggested to make isoflavones more bioavailable in the human body and to retrieve its maximum health benefits. Intestinal LAB strains have the capability of producing the β-glucosidase enzyme that hydrolyzes β-glycosidic linkages present in the isoflavones and results in the production of aglycone compounds, eventually enhancing their antioxidant and anti-tumor properties [4]. Moreover, LAB -mediated fermentation of soymilk also eliminates several non-digestible oligosaccharides by secreting the α-galactosidase enzyme [6]. LAB fermentation also mediates the production of several bioactive peptides such as antibacterial, antithrombotic, hypotensive, immunomodulatory, mineral binding, and opioid peptides that play a significant role in the prevention of various health disorders [7]. In addition, fermenting soymilk with a suitable LAB strain also improves its sensory attributes by removing undesirable beany flavor [5]. Buttermilk is another very popular and desirable nutritional drink for lactose intolerant individuals, produced as a byproduct from the churning of the unfermented milk cream [8]. Buttermilk is also a rich source of proteins, vitamins, milk salts, and nutritional ingredients [9]. Currently, a wide range of buttermilk variants are available in the market but cannot be considered as health drinks due to added sugar, flavors, and preservatives in them, which can further impose serious health problems. Therefore, the preparation of functional buttermilk and soymilk using a natural flavor and a nutrition-imparting LAB strain is highly recommended to reduce their undesirable flavor and enhance consumer acceptability. L-alanine is an FDA-approved food flavor, low-calorie sweetener, fat substitute, and nutritional supplement [10,11]. It is an ideal ingredient for developing pharma- ceutics to treat hypoglycemia, prostate hypertrophy, liver diseases, and urea cycle disorders [12–14]. It is widely used in fragrances, hair and skin conditioners, and personal care products [15,16]. Moreover, it provides high energy during intensive workouts, thus is added as a supplement to energy drinks to enhance athletic performance [17]. Therefore, an L-alanine producing P. acidilactici BD16 (alaD+) strain is being proposed hereby as a starter culture bacterium for the production of functional buttermilk and soymilk. Fermentation of buttermilk and soymilk was carried out for 24 h and evaluated for variations in their physiochemical and quality attributes. Further, GC-MS-based metabolomic fingerprinting was performed to identify the effect of LAB fermentation on increasing levels of certain desirable metabolites with pharmacological and flavor enhancement properties and reducing undesirable and quality detrimental compounds that impart bitterness, unpleasant flavor, rancidity, and unhealthy attributes to the fermented drinks. 2. Materials and Methods 2.1. Pediococcus acidilactici BD16 (alaD+) Strain and Culture Conditions A synthetic alaD gene encoding alanine dehydrogenase enzyme, which catalyzes the reductive amination of pyruvate to L-alanine, was designed insilico and further cloned into the pLES003 vector and expressed using an auto-inducible P289 promoter (unpublished data). The pLES003 vector is a shuttle vector that can replicate in both Gram-negative E. coli as well as in Gram-positive lactic acid bacteria [18,19]. The sequence of the synthetic alaD gene construct was submitted to the Genbank database under the accession number MT108231. The recombinant pLES003alaD vector was further introduced into a native P. acidilactici BD16 (MTCC 10973) strain using standard DNA transformation procedures as mentioned earlier by Kaur and co-workers [18]. The P. acidilactici BD16 (alaD+) harboring pLES003alaD vector was further revived in de Man’s Rogosa Sharpe Medium (MRS) broth containing dextrose 20 g/L, beef extract 10 g/L, peptone 10 g/L, sodium acetate 5 g/L, yeast extract 5 g/L, tri-ammonium citrate 2 g/L, dipotassium hydrogen phosphate 2 g/L, magnesium sulfate 0.1 g/L, manganous sulfate 0.05 g/L, erythromycin 20 µg/mL, and Tween 80 1 mL/L, pH 6.5 ± 0.2 undermicroaerophilic and stationary conditions at 37 ◦C for 24 h [20]. After revival, 2% v/v culture broth grown overnight (containing 106 cfu/mL) was used as an inoculum for the fermentation experiments. Antibiotic pressure was Molecules 2021, 26, 4671 3 of 25 maintained during the revival and subculturing of the recombinant P. acidilactici (alaD+) bearing pLES003alaD vector to maintain its copy number. Keeping in view the food safety aspects, antibiotic addition was avoided during further experiments such as inoculum preparation and fermentation of the drinks. 2.2. Procurement of Buttermilk and Preparation of Soymilk Samples Buttermilk without added flavor and preservatives was procured from Verka Milk Plant, Patiala, Punjab, India. For the preparation of fresh soymilk, an adequate number of whole soybeans

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